Blockade of Glucocorticoid-Induced Tumor Necrosis Factor–Receptor-Related Protein Signaling Ameliorates Murine Collagen-Induced Arthritis by Modulating Follicular Helper T Cells  Jie Ma, Dingqi Feng, Yancai Wei, Jie Tian, Xinyi Tang, Ke Rui, Liwei Lu, Huaxi Xu, Shengjun Wang  The American Journal of Pathology  Volume 186, Issue 6, Pages 1559-1567 (June 2016) DOI: 10.1016/j.ajpath.2016.02.010 Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 1 Up-regulated glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR) expression in CD4+ follicular helper T (Tfh) cells compared with non-Tfh cells in type II collagen (CII)-immunized mice. Flow cytometry analysis of GITR expression in Tfh cells and non-Tfh cells from the spleens of collagen-induced arthritis (CIA) mice. DBA/1J mice were immunized with complete Freund's adjuvant plus CII. Purified CD4+ T cells from the spleens were stained for CXC receptor type 5 (CXCR5), inducible costimulator (ICOS), and GITR and analyzed by flow cytometry. n >3 independent experiments. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 2 Increased frequency and number of CD4+ follicular helper T (Tfh) cells that received glucocorticoid-induced tumor necrosis factor–receptor-related protein ligand (GITRL) treatment in vitro. Splenic CD4+ T cells from normal DBA/1J mice were purified by MACS beads and cultured with different concentrations of GITRL (0.1, 1.0, and 10.0 μg/mL) or control protein treatment. A: The frequencies of Tfh cells in cultures with different treatments are indicated in the representative flow cytometry profiles. B: The total number of Tfh cells in the culture. The expression levels of IL-21 (C) and Bcl-6 (D) mRNA in cultured CD4+ T cells were determined by quantitative RT-PCR analysis. The relative mRNA expression levels of IL-21 or Bcl-6/β-actin from three independent experiments are presented. E: The concentration of IL-21 in the supernatant of T-cell cultures was determined by enzyme-linked immunosorbent assay. The results are representative of three independent experiments. Data are expressed as means ± SD. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 3 Glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR)-Fc protein decreases the severity of the disease in collagen-induced arthritis (CIA) mice. A total of 100 g of bovine type II collagen dissolved in 0.1 mol/L acetic acid was emulsified with an equal volume of complete Freund's adjuvant and administered intradermally at the base of the tail into DBA/1J mice. On day 21, a booster emulsion was administered intradermally. Following the same protocol, adjuvant-treated littermates that were given PBS in place of CII served as controls. Starting from day 22, GITRL protein, GITR-Fc protein, and control protein were injected into the tail vein of the mice (100 μg/mice) every 2 days. A: The development of arthritis among the immunized mice treated with PBS (PBS-CIA), control protein (CTL-CIA), GITRL (GITRL-CIA), and GITR- Fc (GITR-Fc-CIA) was monitored every 2 days. B: The clinical scores of arthritis severity were assessed in the PBS-CIA, CTL-CIA, GITRL-CIA, and GITR-Fc-CIA groups. C: The histopathologic scores of the joint tissue from the PBS-CIA, CTL-CIA, GITRL-CIA, and GITR-Fc-CIA groups. The values are expressed as the means ± SD. D: The joints were sectioned for H&E staining. n = 20 per group (A and B); n = 6 per group (C). ∗P < 0.05, ∗∗P < 0.01. Original magnification, ×100. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 4 Decreased CD4+ follicular helper T (Tfh) cell number in glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR)-Fc protein-treated collagen-induced arthritis (CIA) mice. A: The frequency of splenic CD4+ CXC receptor type 5 (CXCR5)+ inducible costimulator (ICOS)high cells was analyzed by flow cytometry. The percentages of CD4+CXCR5+ICOShigh cells are indicated in the representative flow cytometry profiles (boxed areas). B: The percentage of CD4+CXCR5+ICOShigh cells in the spleens from the four treatment groups. C: The percentage of CD4+CXCR5+ICOShigh cells in the lymph node (LN) from the four treatment groups. D: An immunofluorescence analysis of CD4+CXCR5+ cells in the spleens from the four treatment groups. The spleens of the mice were dissected 42 days after the first immunization. Staining was performed with anti-CXCR5 (green) and anti-CD4 (red) monoclonal antibody, and the combined staining is shown in yellow. The spleens from the GITR ligand (GITRL)-CIA group showed more CD4+CXCR5+ cells compared with the spleens from the CTL-CIA and PBS-CIA groups. The relative expression levels of IL-21 mRNA from cells in the spleen (E) and Bcl-6 mRNA from the CD4+ T cells in the spleen (F) were assessed by real-time quantitative PCR. The relative expression of IL-21 or Bcl-6/β-actin is presented as the means ± SD. G: The concentrations of IL-21 in the serum from immunized mice were determined by enzyme-linked immunosorbent assay. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Original magnification, ×200. CTL, control; CTL-CIA, mice treated with control protein; GITRL-CIA, mice treated with GITRL; PBS, phosphate-buffered saline; PBS-CIA, mice treated with PBS; GITR-Fc-CIA, mice treated with GITR-Fc. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Figure 5 Decreased antibody production in the glucocorticoid-induced tumor necrosis factor–receptor-related protein (GITR)-Fc protein-treated collagen-induced arthritis (CIA) mice. The percentage (A) and number (B) of CD19+ cells in the spleens from the immunized mice. The percentage of CD138+ cells in the spleens (C) and bone marrow (BM; D) of the immunized mice. E: An immunohistochemistry analysis of the GC in the spleen of the immunized mice. The spleen was dissected out of the mice 42 days after the first immunization. F: Anti-CII–specific antibodies (IgG) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. G: Anti-CII–specific antibodies (IgG1) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. H: Anti-CII–specific antibodies (IgG2a) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. I: Anti-CII–specific antibodies (IgG2b) were measured in the sera from the four treatment groups by enzyme-linked immunosorbent assay. J: The number of IgG-secreting cells in the bone marrow was analyzed by ELISpot. K: The number of CII-specific IgG-secreting cells in the bone marrow. L: The number of IgG-secreting cells in the spleen. M: The number of CII-specific IgG-secreting cells in the spleen. n = 3 independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Original magnification, ×200. CII, type II collagen; CTL, control; CTL-CIA, mice treated with control protein; GITR-Fc-CIA, mice treated with GITR-Fc; GITRL-CIA, mice treated with GITRL; OD, optical density; PBS, phosphate-buffered saline; PBS-CIA, mice treated with PBS; SP, spleen. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Supplemental Figure S1 Effects of glucocorticoid-induced tumor necrosis factor–receptor-related protein ligand (GITRL) on proliferation of CD4+ CXC receptor type 5 (CXCR5)+ T cells. Splenic CD4+CXCR5+ T cells were purified from mice spleen cell suspensions by MACS. After staining with carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE) the cells were treated with either 0.1 to 10.0 μg/mL GITRL protein or control protein for 72 hours. Representative flow plots of cells with CFSE are shown on the x axis, and count is shown on the y axis. n = 3 independent experiments. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions

Supplemental Figure S2 Effects of glucocorticoid-induced tumor necrosis factor–receptor-related protein ligand (GITRL) on apoptosis of CD4+ CXC receptor type 5 (CXCR5)+ T cells. Splenic CD4+ T cells were purified from mice spleen cell suspensions by MACS and then treated with 0.2 μg/mL dexamethasone (DEX), in the presence of either 1.0 to 10 μg/mL GITRL protein or control protein for 6 hours. A: Representative flow plots of cells with Annexin V on the x axis and propidium iodide on the y axis. Lower left shows normal cells, lower right, early apoptotic cells, and upper right, late apoptotic and necrotic cells. B: Percentage of early apoptotic cells in different groups. n = 3 independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001. The American Journal of Pathology 2016 186, 1559-1567DOI: (10.1016/j.ajpath.2016.02.010) Copyright © 2016 American Society for Investigative Pathology Terms and Conditions